In humans, defects in protein folding can lead to neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease and debilitating forms of ataxia. The 70 kD heat-shock proteins (Hsp70) are a ubiquitous family of protein chaperones involved in all aspects of protein homeostasis including protein biogenesis, repair and degradation. Hsp70 activity is governed by two types of cofactors; J domain-containing proteins activate the ATP hydrolysis step leading to high affinity substrate binding, while nucleotide exchange factors (NEFs) promote ADP dissociation and polypeptide release. Eukaryotes from yeast to humans express three recently described families of structurally diverse cytosolic NEFs (Hsp110 (Sse1/2 in yeast), HspBP1 (Fes1), and Bag-domain (Snl1)) that perform essentially the same biochemical function. The distinct contributions these proteins make to Hsp70-dependent activities are unknown in any biological system. The primary goal of this proposal is to determine how these NEFs partner with cytosolic Hsp70 chaperones to mediate proteostasis in eukaryotic cells. We hypothesize that while the NEFs share a common ability to stimulate Hsp70 activity, they differentially interact with the Ssa and Ssb families of cytosolic Hsp70 to promote protein folding and repair. In addition, we hypothesize that the Bag homolog Snl1 simultaneously recruits Hsp70 and the ribosome to promote protein biogenesis at the endoplasmic reticulum (ER). Three lines of investigation are proposed to test these hypotheses. In the first aim, we will determine how the apparently principal NEF Hsp110 is integrated into the cytosolic NEF network to promote protein biogenesis and repair. A major component of this aim will be genetic and cell biological experiments to assess the contribution of the Hsp110 substrate binding domain to Hsp70- dependent protein folding in vivo. In contrast to published results that Fes1 binds Ssa and Ssb in vitro, we have obtained preliminary evidence that Fes1 interacts solely with Ssa in vivo. In Aim 2 we will resolve these conflicting findings and determine the mechanism of Fes1-Hsp70 specificity. Lastly, we have discovered that the ER membrane-associated Snl1 binds the ribosome in addition to Hsp70. Aim 3 will be focused on determining the physiological significance of membrane recruitment of the translation machinery by an Hsp70 NEF. These studies represent the first comprehensive analysis of cytosolic Hsp70 NEFs by genetic, biochemical, and genomic approaches. Due to the high degree of conservation of these and other components of the Hsp70 chaperone network in eukaryotes, the results will be directly applicable to understanding the roles of Hsp70 NEFs in protein biogenesis and quality control in human cells.